1. Field of the Invention
This invention relates to the methods and compositions suitable for the detection, analysis, quantitation and sequencing of double stranded nucleic acids.
2. Description of the Related Art
A. Invasion of double stranded nucleic acid
Linear, non-supercoiled double-stranded DNA (dsDNA) is known to be able to accommodate an additional oligonucleotide strand with much less efficiency as compared with single-stranded nucleic adds and supercoiled DNAs. Formation of intermolecular triplexes is mostly limited to long homopurine-homopyrimidine regions (See: Frank-Kamenetskii, M. D., and Mirkin, S. M. (1995) Annu. Rev. Biochem. 64, 65-95 and Soyfer, V. N. and Potaman, V. N. (1996) Triple-Helical Nucleic Acids (Springer, New York). D-loops are formed in linear dsDNA only at the ends of the DNA duplex and when using long single-stranded DNA molecules (See: Wetmur, J. G. (1991) Critical Rev. Biochem. Mol. Biol. 26, 227-259). R-loops may be formed inside linear dsDNA, but long RNAs and transient DNA denaturation is required (See: Thomas, M., White, R. L., and Davis, R. W. (1976) Proc. Natl. Acad. Sci. USA 73, 2294-2298). A complex between an oligodeoxynucleotide (ODN) and linear dsDNA can be formed with the assistance of the RecA protein. However, the fidelity of recognition of this complex is lower as compared with the protein-free DNAxe2x80x94DNA. Moreover, the complex is unstable upon deproteinization (See: West, S. C. (1992) Annu. Rev. Biochem. 61, 603-640 and Malkov, V. A., Sastry, L. and Camerini-Otero, R. D. (1997) J. Mol. Biol. 271, 168-177). It has recently been demonstrated that a pair of complementary modified ODNs will bind to dsDNA as a result of their self-mediated invasion of the DNA duplex. However, these complexes were formed only at the ends of linear dsDNA (See: Kutyavin, I. V., Rhinehart, R. L., Lukhtanov, E. A., Gorn, V. V., Meyer, R. B., Jr., and Gamper, H. B., Jr. (1996) Biochemistry 35, 11170-11176). In addition, a few techniques exist for the formation of specific complexes between ODNs and dsDNA based upon either prior DNA denaturation or degradation of one DNA strand before ODN binding. These techniques, however, require subsequent reconstruction or reparation of the DNA duplex (See: Shepard, A. R., and Rae, J. L. (1997) Nucleic Acid Res. 25, 3183-3185 and Anonymous (1997/1998) in Gibco BRL Products and Reference Guide, (Life Technologies, Gaithersburg, Md.), pp. 1914-1915).
B. Sequencing Double Stranded Nucleic Acids
Progress in enzymatic (or dideoxy) DNA sequencing has (See: Sanger, F., Nicklen, S. and Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467) completely changed the science of molecular genetics and revolutionized the field of modern biotechnology. However, the development of improved dideoxy sequencing methodologies as well as the introduction of new sequencing approaches promises to further facilitate great advancements in science.
High quality sequence data is generally obtained using dideoxy sequencing reactions on purified single-stranded (ss) DNA templates. Consequently, Sanger sequence typically requires the performance of laborious ssDNA isolation (See: Griffin, H. G. and Griffin, A. M., eds. (1993) DNA Sequencing Protocols. Humana Press, Totowa, N.J., USA, Brown, T. A. (1994) DNA Sequencing: The Basics. IRL Press, Oxford, GB and Ansorge, W., Voss, H. and Zimmermann, J., eds. (1997) DNA Sequencing Strategies: Automated and Advanced Approaches. Wiley, New York, N.Y., US). To avoid ssDNA isolation, direct sequencing of double-stranded (ds) DNA was developed. However, the robustness of direct sequencing denatured dsDNA is often compromised by poor sequence readability and/or spurious sequence data resulting from non-specific mispriming. For this reason, isothermal dideoxy sequencing of dsDNA is normally limited to constructs of less than 50 kb. Thermal cycle sequencing overcomes this size limitation thereby allowing multimegabase-template dsDNA to be directly sequenced (See: Heiner, C. R., Hunkapiller, K L., Chen, S. -M., Glass, J. I. and Chen, E. Y. (1998) Genome Res. 8, 557-561). However, careful choice of various parameters and operation with the thermal cycler is requisite for cycle sequencing.
Therefore, it is highly desirable to develop isothermal methods for sequencing non-denatured dsDNA. To this end, solid phase sequencing of dsDNA restriction fragments by strand displacement or nick translation was recently described (See: Fu, D. -J., Kxc3x6ster, H., Smith, C. L. and Cantor, C. R. (1997) Nucleic Acids Res. 25, 677-679). Still, this approach cannot be applied for direct sequencing of long DNA or closed circular dsDNA.
C. Nucleic Acid Comprising Topologically Linked Structures
DNA is well known to adopt various topological (and pseudotopological) structures like knots, catenanes, Borromean rings and pseudorotaxanes. (See: M. D. Frank-Kamenetskii, J. Mol. Struct. (Theochem) 1995, 336, 235-243; N. C. Seeman, Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 225-248; K. Ryan, E. T. Kool, Chem. and Biol. 1998, 5, 59-67; N. C. Seeman, Angew. Chem. 1998, 110, 3408-3428; and Angew. Chem. Int. Ed. Engl. 1998, 37, 3220-3238). It has long been recognized that DNA topology plays a crucial role in such fundamental biological phenomena as DNA supercoiling and topoisomerization (See M. D. Frank-Kamenetskii, Unraveling DNA: The most important molecule of life, Addison-Wesley, Reading, Mass., USA 1997, p. 214; and R. Sinden, DNA Structure and Function, Academic Press, San Diego, Calif., USA 1994, p. 398). Another reason for a considerable interest in higher order DNA topology structures stems from the realization that DNA topological and pseudotopological forms may provide stable and sequence-specific targeting of DNA. Accordingly, highly localized DNA detection and precise spatial positioning of various ligands on DNA scaffold becomes possible. This may lead to new applications in molecular biotechnology, gene therapy and in the emerging field of DNA nanotechnology (See: N. C. Seeman, Acc. Chem. Res. 1997, 30, 357-363; b) C. M. Niemeyer, Angew. Chem. 1997, 109, 603-606; and Angew. Chem. Int. Ed. Engl. 1997, 36, 585-587).
One of promising DNA pseudotopological constructions is the DNA padlock consisting of a long single-stranded (ss) DNA molecule forming a pseudorotaxane with a short cyclic oligodeoxynucleotide (cODN) (See: M. Nilsson, H. Malmgren, M. Samiotaki, M. Kwiatkowski, B. P. Chowdhary, U. Landegren, Science 1994, 265, 2085-2088; M. Nilsson, K. Krejci, J. Koch, M. Kwiatkowski, P. Gustavsson, U. Landegren, Nature Gen. 1997, 16, 252-254; P. M. Lizardi, X. Huang, Z. Zhu, P. Bray-Ward, D. C. Thomas, D. C. Ward, Nature Gen. 1998, 19, 225-232; and J. Banxc3xa9r, M. Nilsson, M. Mendel-Hartvig, U. Landegren, Nucleic Acids Res. 1998, 26, 5073-5078). Another interesting pseudorotaxane-type structure is the sliding clamp which contains a short cODN threaded on double-stranded (ds) DNA (See: K. Ryan, E. T. Kool, Chem. and Biol. 1998,5, 59-67, d) N. C. Seeman, Angew. Chem. 1998, 110, 3408-3428). Notwithstanding the value of the indicated pseudotopological structures for DNA labeling, note that in these constructions the cODN tag is allowed to slide along the target for considerable distances thereby compromising the precision of spatial positioning of the label.
Generally this invention relates to methods and compositions pertaining to PD-Loops. In one embodiment, this invention relates to a composition comprising a double stranded nucleic acid having at least one homopurine site and one or more PNA oligomers (at times the PNA oligomers will be referred to herein as xe2x80x9copenersxe2x80x9d) which hybridize to the one or more homopurine sites to thereby create an extended open region inside the double stranded nucleic acid. To the extended open region of the double stranded nucleic acid is then hybridized a nucleobase polymer. The resulting novel composition is a PD-Loop.
In another embodiment, this invention relates to a method for hybridizing a nucleobase polymer to a double stranded nucleic acid to thereby form a PD-Loop. According to the method, a double stranded nucleic add comprising at least one homopurine site is chosen. To the one or more homopurine sites are then hybridized one or more PNA oligomers to thereby create an extended open region inside the double stranded nucleic acid. To this extended open region is hybridized a nucleobase polymer. This method for hybridizing a nucleobase polymer to a double stranded nucleic acid is unique and useful since the duplex need not be chemically or thermally denatured.
In another embodiment of this invention, the PD-Loop can be used to generate Sanger sequence ladders suitable for sequence analysis of the double stranded nucleic acid. In this embodiment, the nucleobase probe is a primer. According to the method, the PD-Loop is formed as previously described and then primer extension is initiated under suitable Sanger sequencing conditions. Under isothermal conditions, this process generates Sanger sequencing ladders from the double stranded nucleic acid template. The Sanger sequencing ladders can then be analyzed by conventional techniques to thereby determine the sequence of the double stranded nucleic add. The method can be repeated until no more suitable homopurine sites are found which would allow one to form a PD-Loop, until the entire sequence of the double stranded template is determined or until the desired sequence information is obtained.
In still another embodiment, this invention pertains to a Sanger sequence ladder which is generated isothermally from a double stranded nucleic acid without the application of chemical or thermal denaturing conditions.
In still another embodiment, this invention is related to a double stranded nucleic acid having a linked single stranded closed circular nucleic acid wherein the single stranded nucleic acid is threaded through the strands of the duplex. This construct will at times be referred to herein as the xe2x80x9cEarringxe2x80x9d.
The invention further relates to a method of forming a double stranded nucleic acid having a linked single stranded closed circular nucleic acid wherein the single stranded nucleic acid is threaded through the strands of the duplex. According to the method, the double stranded nucleic acid is invaded to thereby create an extended open region inside the double stranded nucleic acid. To the extended open region is then hybridized an oligonucleotide in such a way that the two termini of the oligonucleotide are complementary to the exposed double stranded nucleic acid and are juxtapositioned to one strand of the double stranded nucleic acid. Once this PD-Loop is formed, the two termini of the oligonucleotide are then ligated to thereby form the single stranded closed circular nucleic acid. Preferably, the termini are ligated using a ligase but optionally the termini can be ligated using chemical methodology.
In yet another embodiment, the single stranded closed circular nucleic acid is used in a signal amplification methodology. According to the method, a primer is hybridized to the single stranded closed circular nucleic acid. A polymerase dependent primer extension reaction is then initiated to thereby generate one or more single stranded copies of the single stranded dosed circular nucleic acid. Preferably, numerous copies of the single stranded closed circular nucleic acid will be produced such that it results in efficient signal amplification by the detection of the copy or copies. In the most preferred embodiment, a hybridization site for a reporter probe is repeated numerous times per generated copy of the single stranded closed circular nucleic acid.